The products of erythrocyte lyses, hemoglobin (Hb) and heme, are recognized as neurotoxins and the main contributors to delayed cerebral edema and tissue damage after intracerebral hemorrhage (ICH). Finding a means to efficiently control the detoxification and absorption of the hemolytic products (Hb and heme) is an important clinical challenge and critical for hematoma resolution. Clearance of cell-free Hb/heme through phagocytosis/endocytosis is an essential function of microglia/macrophages in anti-inflammatory response. After ICH, the cell-free Hb and heme released from erythrocytes are first bound to haptoglobin (Hp) and hemopexin (Hx), forming stable Hb-Hp and heme-Hx complexes which are subsequently endocytosed by microglia/macrophages through the Hb scavenger receptor CD163 and the heme scavenger receptor CD91, respectively. Our pilot studies and existing data suggest that Hp and Hx expression is regulated by Nrf2, whereas CD163 and CD91 expression is promoted by macrophage colony-stimulating factor (M-CSF) or Dexamethasone (DEX). We propose that efficient detoxification and removal of Hb and heme from the brain require the coordinated effort of all the components for this Hb/heme clearing system. Hence, our central hypothesis is that the endogenous Hb/heme detoxifying/clearing system (CD163-Hp-Hb and CD91-Hx-heme) is essential for mitigating brain damage mediated by erythrocyte- lysis products after ICH. Activating this endogenous Hb/heme cleaning system by upregulating the expression of Hp/Hx and CD163/CD91 through pharmacological approaches (by SF and by M-CSF or DEX) may accelerate the resolution of hemolytic products, thereby counteracting oxidative injury and mitigating the inflammatory response after ICH. To test our hypothesis, we will employ pharmacological and molecular approaches, including gene knockdown/knockout (KO) and overexpression of Hp, Hx, CD163 and CD91 in a primary brain cell culture system and an animal model of ICH through two specific aims (SA). SA1 will explore the role of CD163 and CD91 as scavenger receptors in mediating the endocytosis of Hb and heme that was neutralized by Hp and Hx by using the CD163- and CD91-positive microglia/monocytes and the primary neuron and glia cultures prepared from Hp-KO, Hp-Tg, Hx-KO, Hx-Hp-dKO (double KO). Additionally, we will explore the regulation of CD163 and CD91 by M-CSF or DEX in the primary microglia/macrophage cultures and the regulation of Hp and Hx by Nrf2 by using the primary neuron and glia cultures from Nrf2-KO mice. Next, we will optimize the strategy for efficient endocytosis of hemolytic products and for cytoprotection via combining SF with M-CSF or DEX in an ICH-like injury model in primary neuron-glial co-cultures prepared from wild-type mice. SA2 will evaluate the protective role of Hp and Hx in ICH by subjecting Hp-KO, Hx-KO, Hp-Hx-dKO, Hp-Tg and wild type mice to a modified model of ICH. We will also test the strategy for the optimized removal of Hb/heme by treatment with SFCSF in an autologous blood injection model of ICH in the wild type mice. We believe that our study will help to better understand the mechanism controlling the hematoma resolution. Our long-term goal is to develop a new clinical strategy for treatment of ICH based on enhanced function of the endogenous/innate scavenger system.